The proposed research addresses key unanswered questions about the structures and events by which antigen-activated immunoreceptors initiate cellular signaling leading to diverse responses. The high affinity receptor for lgE (FcepsilonRI) on RBL-2H3 mast cells serves as a valuable model system, and new experimental thrusts will build on previous extensive studies. Specific Aim #1 extends experiments with structurally defined ligands to extract critical features of ligand architecture and cross-linking mechanism that facilitate or inhibit signaling responses. Specific Aim #2 continues efforts to understand the molecular basis by which lipid rafts participate in FcepsilonRI signal initiation, and to achieve functional reconstitution of this receptor in model membranes from purified, defined components. Specific Aim #3 proposes real-time imaging studies to elucidate the process by which FcepsilonRI becomes segregated from particular lipid raft components, and investigate how plasma membrane locations for stimulated exocytosis are determined. Specific Aim #4 proposes experiments to understand the mechanisms by which Rho family proteins and other low molecular weight GTPases participate in early FcepsilonRI signaling, as revealed by previous studies with mutant RBL-B6A4C1 mast cells. These investigations integrate diverse physical, chemical, and biological approaches to provide novel insights into the structure and molecular mechanisms of IgE-FcepsilonRI signaling, and they will advance our molecular understanding of the cell biology of this important immune cell prototype. [unreadable] [unreadable] [unreadable] [unreadable]